Fluorometric system, method and test article

Abstract

A fluorometric system to determine the kind and amount of substances derived from a biological fluid (e.g., serum or urine) or tissue in which the substances to be detected (e.g., antigen, antibody, hormone or enzyme) are coated onto a substrate surface in fluorescent form. Multiple coating areas of different samples may be employed. The fluorometric system includes a source of filtered light to excite fluorescence, optical systems for conducting the excitation light to such coating, and optical systems for receiving emitted fluorescence and for detecting the same. The system efficiency and optical characteristics disclosed avoid photo-bleaching; limit fading; and are especially adapted to provide accurate surface reading fluorometry.

What is claimed is: 
     
       1. In apparatus for analyzing a sample by fluorometric techniques, a carrier member having a surface portion adapted for receiving said sample and including means for supporting said sample thereon as an exposed layer presenting a surface for analysis, test stage means for supporting said member at an analysis location in said apparatus, said carrier member and said stage means being constructed and arranged so that said member is removably insertable into said stage means,   illumination means including a source of excitation radiation and means forming a first optical system intersecting said analysis location for delivering said radiation to the surface thereat,   optical fluorescence collection means including a photodetector and means forming a second optical system intersecting said analysis location for receiving the fluorescence emitted from the exposed layer thereat,   emission filter means associated with said fluorescence collection means for restricting the sensitivity thereof to a frequency band overlapping the emission spectra of said fluorometrically active substance and being substantially non-responsive to the band of excitation radiation,   means juxtaposed with said excitation filter means for forming a beam splitter for removing a portion of the light therefrom for developing a reference channel in parallel with the illumination means,   means for alternating said illumination means and said reference means during mutually exclusive periods of time,   sensing means for developing a signal indicative of sample illumination on-times when said photodetector is activated by sample fluorescence and for developing a second signal indicative of the period when said reference means is detected,   a demultiplexer response to said signal for alternately delivering outputs indicative of said sample signal or said reference signal,   a sample signal processor for receiving said sample signal,   a reference signal processor for receiving said reference signal,   said processors serving to develop signal outputs, the strength of which is proportional to the amount of light signal received at said photodetector during said sample and reference periods, respectively,   a ratio circuit for obtaining the ratio S/R where S equals sample signal and R equals said reference signal, and   display means responsive to said ratio circuit for displaying the value of said ratio.   
     
     
       2. An apparatus as in claim 1 together with excitation filter means associated with said illumination means for limiting the excitation radiation to a predetermined frequency band overlapping the absorption band of said fluorometrically active substance and being substantially non-emissive in the band of fluorometric emission of said substance. 
     
     
       3. An apparatus as in claim 1 together with means disposed in said reference means for forming a diffuse screen therein, and means for collecting light from a portion of said diffuse screen and for inserting the same into said second optical system subsequent to said emission filter means. 
     
     
       4. An apparatus as in claim 1 in which said display means displays the numerical value of said ratio in digital form. 
     
     
       5. An apparatus as in claim 1 in which said alternating means comprises means for alternately blocking and opening said illumination means and said reference means. 
     
     
       6. In a fluorometric testing apparatus in which a sample is tested by illuminating the same with excitation radiation of a predetermined frequency band which it absorbs and re-emits in a second predetermined frequency band, said first frequency band being of higher energy than said second band, emission filter means associated with a fluorescence collection system for restricting the sensitivity thereof to a frequency band overlapping the emission spectra of said fluorometrically active sample and being substantially non-responsive in the band of excitation radiation, said emission filter means comprising a multi-cavity interference filter including an emission frequency pass band filter in combination with a cut-on filter for eliminating wavelengths shorter than said emission spectra and transmissive at frequencies thereabove. 
     
     
       7. A fluorometric testing apparatus as in claim 6 in which said emission filter is constructed by depositing interference coatings on a substrate, said pass band filters and said cut-on filter being disposed on the same substrate. 
     
     
       8. A fluorometric apparatus as in claim 6 in which said sample is tagged with dye fluorescein isothiocyanate having an adsorption band at approximately 480nm and an emission band at approximately 530 nm, said emission filter being of the interference type and comprising a three-cavity, layered structure having a pass band wavelength of 540nm, and interference filter means forming a cut-on filter having a pass frequency at 550 nm with transmission of 80% thereat, said combination filter having a rejection to response in the band of excitation radiation of at least 5 order of magnitude. 
     
     
       9. An apparatus as in claim 6 together with excitation filter means associated with said apparatus for limiting the excitation radiation delivered to the sample to a predetermined frequency band overlapping the absorption band of said sample and being substantially non-emissive in the band of fluorometric re-emission from said sample comprising a multi-cavity interference filter including excitation frequency pass band filter having a maximum transmission at a predetermined wavelength in combination with a cut-off filter which eliminates wavelengths in a frequency band longer than said excitation frequencies. 
     
     
       10. Apparatus as in claim 9 in which said first filter means is constructed of a multi-cavity interference filter and in which said excitation pass band filter and said cut-off filter are disposed on the same substrate.